Design syntheses and mitochondrial complex I inhibitory activity of novel acetogenin mimics.

Some natural acetogenins are the most potent inhibitors of mitochondrial complex I. These compounds are characterized by two functional units [i.e. hydroxylated tetrahydrofuran (THF) and alpha, beta-unsaturated gamma-lactone ring moieties] separated by a long alkyl spacer. To elucidate which structural factors of acetogenins, including their active conformation, are crucial for the potent inhibitory activity we synthesized a novel bis-acetogenin and its analogues possessing two gamma-lactone rings connected to bis-THF rings by flexible alkyl spacers. The inhibitory potency of the bis-acetogenin with bovine heart mitochondrial complex I was identical to that of bullatacin, one of the most potent natural acetogenins. This result indicated that one molecule of the bis-acetogenin does not work as two reactive inhibitors, suggesting that a gamma-lactone and the THF ring moieties act in a cooperative manner on the enzyme. In support of this, either of the two ring moieties synthesized individually showed no or very weak inhibitory effects. Moreover, combined use of the two ring moieties at various molar ratios exhibited no synergistic enhancement of the inhibitory potency. These observations indicate that both functional units work efficiently only when they are directly linked by a flexible alkyl spacer. Therefore, some specific conformation of the spacer must be important for optimal positioning of the two units in the enzyme. Furthermore, the alpha,beta-unsaturated gamma-lactone, the 4-OH group in the spacer region, the long alkyl tail attached to the THF unit and the stereochemistry surrounding the hydroxylated bis-THF rings were not crucial for the activity, although these are the most common structural features of natural acetogenins. The present study provided useful guiding principles not only for simplification of complicated acetogenin structure, but also for further wide structural modifications of these molecules.     

Structure-activity relationship of chemical defenses from the freshwater plant Micranthemum umbrosum

Vascular plants produce a variety of molecules of phenylpropanoid biosynthetic origin, including lignoids. Recent investigations indicated that in freshwater plants, some of these natural products function as chemical defenses against generalist consumers such as crayfish. Certain structural features are shared among several of these anti-herbivore compounds, including phenolic, methoxy, methylenedioxy, and lactone functional groups. To test the relative importance of various functional groups in contributing to the feeding deterrence of phenylpropanoid-based natural products, we compared the feeding behavior of crayfish offered artificial diets containing analogs of elemicin (1) and beta-apopicropodophyllin (2), chemical defenses of the freshwater macrophyte Micranthemum umbrosum. Both allyl and methoxy moieties of 1 contributed to feeding deterrence. Disruption of the lactone moiety of 2 reduced its deterrence. Finally, feeding assays testing effects of 1 and 2 at multiple concentrations established that these two natural products interact additively in deterring crayfish feeding.     

beta-D-Glucose 1-phosphate. A structural unit and an immunological determinant of a glycan from streptococcal cell walls

Glycose 1-phosphate moieties are emerging as important structural units of macromolecular substances imparting special biological functions to these molecules. In the present study, beta-D-glucose 1-phosphate moieties are shown to be structural units and immunological determinants of a bacterial glycan. The glycan is a tetraheteroglycan from the cell wall of Streptococcus faecalis, strain N and is composed of glucose, galactose, rhamnose, N-acetylgalactosamine, and phosphate. Several lines of evidence have been obtained for the presence of beta-D-glucose 1-phosphate units in the glycan, including the liberation of glucose by mild acid hydrolysis, the inhibition of the precipitin reaction by beta-D-glucose 1-phosphate, and the formation of levoglucosan on treatment of the glycan with alkali. Work on the preparation of affinity adsorbents for isolating the new types of antibodies directed at the beta-D-glucose 1-phosphate moieties is in progress.     

Synthesis of water-soluble phthalocyanines bearing four or eight d-galactose units

The synthesis and structural characterization of two glycophthalocyanines with four or eight unprotected d-galactose units is reported. The sugar units are linked to the macrocycle via the hydroxyl group located at C-6. The water solubility promoted by the carbohydrate moieties provides a potential application of these phthalocyanine derivatives as photosensitizers in photodynamic therapy.     

Characterization of an acetylated heteroxylan from Eucalyptus globulus Labill

A heteroxylan was isolated from Eucalyptus globulus wood by extraction of peracetic acid delignified holocellulose with dimethyl sulfoxide. Besides (1-->4)-linked beta-D-xylopyranosyl units of the backbone and short side chains of terminal (1-->2)-linked 4-O-methyl-alpha-D-glucuronosyl residues (MeGlcA) in a 1:10 molar ratio, this hemicellulose contained galactosyl and glucosyl units attached at O-2 of MeGlcA originating from rhamnoarabinogalactan and glucan backbones, respectively. About 30% of MeGlcA units were branched at O-2. The O-acetyl-(4-O-methylglucurono)xylan showed an acetylation degree of 0.61, as determined by 1H NMR spectroscopy, and a weight-average molecular weight (M(w)) of about 36 kDa (P=1.05) as revealed from size-exclusion chromatography (SEC) analysis. About half of the beta-D-xylopyranosyl units of the backbone were found as acetylated moieties at O-3 (34 mol%), O-2 (15 mol%) or O-2,3 (6 mol%). Practically, all beta-D-xylopyranosyl units linked at O-2 with MeGlcA residues were 3-O-acetylated (10 mol%).     

Exploring overlapping functional units with various structure in protein interaction networks

Revealing functional units in protein-protein interaction (PPI) networks are important for understanding cellular functional organization. Current algorithms for identifying functional units mainly focus on cohesive protein complexes which have more internal interactions than external interactions. Most of these approaches do not handle overlaps among complexes since they usually allow a protein to belong to only one complex. Moreover, recent studies have shown that other non-cohesive structural functional units beyond complexes also exist in PPI networks. Thus previous algorithms that just focus on non-overlapping cohesive complexes are not able to present the biological reality fully. Here, we develop a new regularized sparse random graph model (RSRGM) to explore overlapping and various structural functional units in PPI networks. RSRGM is principally dominated by two model parameters. One is used to define the functional units as groups of proteins that have similar patterns of connections to others, which allows RSRGM to detect non-cohesive structural functional units. The other one is used to represent the degree of proteins belonging to the units, which supports a protein belonging to more than one revealed unit. We also propose a regularizer to control the smoothness between the estimators of these two parameters. Experimental results on four S. cerevisiae PPI networks show that the performance of RSRGM on detecting cohesive complexes and overlapping complexes is superior to that of previous competing algorithms. Moreover, RSRGM has the ability to discover biological significant functional units besides complexes.     

Heterogeneity of the interflavanyl bond in proanthocyanidins from natural sources lacking C-4 (C-ring) deoxy flavonoid nucleophiles

The proanthocyanidin pool in the floral kingdom usually involves the presence of carbon-carbon bonds linking predominantly flavan-3-ol constituent moieties. Such an ensemble of flavan-3-ol units originates via electrophilic aromatic substitution of flavan-4-yl carbocations (or their equivalents) derived from flavan-4-ols and/or flavan-3,4-diols and the nucleophilic centers of the m-oxygenated A-rings of flavan-3-ol nucleophiles. In the absence of these potent flavan-3-ol nucleophiles with their aptitude for the formation of carbon-carbon bonds, alternative centers emerge as participants in interflavanyl bond formation. Such a phenomenon is demonstrated for the distribution of various profisetinidin-, prorobinetinidin-, proguibourtinidin-, promelacacinidin- and proteracacinidin-type pro- and leuco-anthocyanidins in several southern hemisphere heartwood species.     

Structural characterization of Schistosoma mansoni adult worm glycosphingolipids reveals pronounced differences with those of cercariae

Immune responses induced by glycans upon infection with Schistosoma mansoni may be mediated by either schistosomal glycoproteins or glycosphingolipids. In this study, we have elucidated the structural features of both carbohydrate moieties and respective ceramide units of complex glycosphingolipids from adult S. mansoni. Obtained data revealed a vast structural heterogeneity due to manifold combinations of different oligosaccharides and ceramide entities. Observed carbohydrate moieties included Lewis(X) (Le(X); Gal(β1-4)[Fuc(α1-3)]GlcNAc) as well as, in part, multiply fucosylated LacdiNAc (LDN; GalNAc(β1-4)GlcNAc) carbohydrate epitopes. Corresponding lipid portions comprised predominantly C18-sphingosine as well as C18- and C20-phytosphingosine derivatives. Intriguingly, glycosphingolipids carrying an Le(X) epitope contained predominantly C18-sphingosine, whereas LDN-based species exhibited mostly phytosphingosine derivatives, in addition to C18-sphingosine, indicating that the two classes of glycosphingolipids might be synthesized via different biosynthetic routes. Compared with literature data, adult worm glycosphingolipids with Le(X) epitopes revealed clear structural differences in comparison to corresponding cercarial species which have been shown to exhibit mainly sphinganine bases with 18-21 carbon atoms. Therefore, it may be hypothesized that the divergent structural features of the respective ceramide moieties are responsible for the published observation that only adult worm, but not cercarial glycosphingolipids are able to induce dendritic cell activation skewing the T-cell response toward a Th1 profile.     

Structural polymorphism in DNA

We have used the enzyme micrococcal nuclease and the methylating reagent dimethyl sulfate to examine the structural properties of eukaryotic DNAs. Our studies demonstrate extensive structural polymorphism in the DNA double helix. Moreover, we find that the distribution of helical variants is in some instances correlated with the functional organization of the DNA. These observations raise the possibility that eukaryotic DNAs may be organized into discrete functional units having characteristic structural properties. In addition, we find that boundaries between different functional units are typically marked by DNA segments having unusual conformational properties. Such structural perturbations could serve as signals in the utilization of genetic information in eukaryotes, and may be important in a variety of different protein-DNA interactions.     

Definition of crucial structural factors of acetogenins, potent inhibitors of mitochondrial complex I

Some natural acetogenins are the most potent inhibitors of bovine heart mitochondrial complex I. These compounds are characterized by two functional units (i.e. hydroxylated tetrahydrofuran (THF) and alpha,beta-unsaturated gamma-lactone ring moieties) separated by a long alkyl spacer. To elucidate which structural factors of acetogenins including their active conformation are crucial for the potent inhibitory effect, we synthesized a series of novel acetogenin analogues possessing bis-THF rings. The present study clearly demonstrated that the natural gamma-lactone ring is not crucial for the potent inhibition, although this moiety is the most common structural unit among a large number of natural acetogenins and has been suggested to be the only reactive species that directly interacts with the enzyme (Shimada et al., Biochemistry 37 (1998) 854-866). The presence of free hydroxy group(s) in the adjacent bis-THF rings was favorable, but not essential, for the potent activity. This was probably because high polarity (or hydrophilicity), rather than hydrogen bond-donating ability, around the bis-THF rings is required to retain the inhibitor in the active conformation. Interestingly, length of the alkyl spacer proved to be a very important structural factor for the potent activity, the optimal length being approximately 13 carbon atoms. The present study provided further strong evidence for the previous proposal (Kuwabara et al., Eur. J. Biochem. 267 (2000) 2538-2546) that the gamma-lactone and THF ring moieties act in a cooperative manner on complex I with the support of some specific conformation of the spacer.     

Structural investigation of the carbohydrate element of human pituitary follicle-stimulating hormone by methylation analysis

A preparation of human follicle-stimulating hormone has been subjected to methylation analysis by using the methyl sulphinyl carbanion-dimethyl sulphoxide-methyl iodide method. The hydrolysis products of the methylated glycoprotein were reduced, acetylated, analysed by gas-phase chromatography and mass spectrometry and identified by comparison with standards. Methylation analysis demonstrated that (1) the d-galactose, mannose, fucose and 2-amino-2-deoxyglucose units exist in the pyranose forms, (2) the 2-amino-2-deoxyglucose units are N-acetylated, (3) the fucopyranose units occupy terminal non-reducing positions, (4) the d-galactopyranose units are linked in the 1- and 2-positions, (5) the mannopyranose units exist in three forms, some as terminal non-reducing residues, some as 1,6-linked residues and some as 1,3,4-linked branch points, and (6) the 2-acetamido deoxyglucopyranose units are 1,6-linked. These structural assignments are compared with other data previously obtained for the carbohydrate moieties of follicle-stimulating hormone.     

Structural analysis of carrageenans from the red alga, Callophyllis hombroniana Mont. Kütz (Kallymeniaceae, Rhodophyta)

The use of a range of modern analytical techniques has facilitated the structural characterisation of the polysaccharide from the New Zealand endemic red alga, Callophyllis hombroniana. The native polysaccharide contains a number of structural units with the largest proportion consisting of 3-linked beta-D-galactopyranosyl 2-sulfate units, alternating with 4-linked 3,6-anhydro-alpha-D-galactopyranosyl 2-sulfate units, that is, theta-carrageenan (36 mol%). C. hombroniana is the first red seaweed reported to naturally contain such a large proportion of theta-carrageenan.     

Seven new steroidal glycosides from the roots of Cynanchum forrestii

Seven new steroidal glycosides, cynaforrosides K (1), L (2), M (3), N (4), and Q (7), based on a 13,14:14,15-disecopregnane-type aglycone, and cynaforrosides O (5) and P (6) with a 14,15-secopregnane-type aglycone, were isolated from the 95% ethanol extract of the roots of Cynanchum forrestii Schlechter. The structures of new compounds were determined on the basis of spectroscopic and chemical evidence. The sugar units of cynaforrosides K-P contained two moieties of glucoses with the mode of 1-->4 linkage and those of cynaforrosides K-O contained six moieties of sugars.     

Preferential Marriage among the Rukuba of Benue-Plateau State, Nigeria

The overall system of Rukuba marriage is formally a straight exchange of women between the component units of two exogamous moieties, component units which in turn abduct each other's wives in secondary marriage. The youths and girls of each of these component units pair off in formalized premarital relationships until girls marry out in the other moiety. The exchange pattern between the moieties works in such a way that the eldest girl of a set of uterine sisters must marry her mother's last lover's son, and her junior sisters are preferentially married in their mother's natal component unit, thus introducing a delayed exchange between the component units of opposite moieties working concurrently with the straight exchange system between the moieties. This is made possible by the various combinations of primary and/or preferential marriage and subsequent secondary marriages. [Rukuba, moieties, Nigeria, preferential marriage]     

Role of the tertiary structure in the diphenol oxidase activity of Octopus vulgaris hemocyanin

The functional differences between the oxygen transport protein Hemocyanin and the enzymes Tyrosinase and Catechol oxidase are believed to be governed, at least in part, by the tertiary structure, which differs in these molecules and controls the accessibility of their copper containing active site for substrate(s). Accordingly, Octopus vulgaris Hemocyanin catalyses the o-diphenol oxidation to o-quinone at a very low rate. The crystallographic structure of one of the functional units (called Odg) of O. dofleini Hemocyanin shows two domains, a mainly α-helical domain that directly binds the copper ions of the reaction center and a β-strand domain that precludes access to the active site to ligands bigger than molecular oxygen. In this work, we have first cleaved the whole protein and then purified different oxygen binding functional units from O. vulgaris Hemocyanin. These functional units were used in activity assays with l-DOPA, the paradigmatic substrate for Catechol oxidase. All functional units show a negligible enzymatic activity. The procedure to generate the functional units induces in only one of them a proteolytic cleavage. Amino terminal sequencing and mass spectroscopy of the fragments allow to place the cleavage site between the alpha and beta domains of the functional unit homologous to Odd, in the O. dofleini sequence. An increase, up to three orders of magnitude, of Tyrosinase-like activity was observed when the cleaved Odd-like was incubated with the substrate in the presence of trifluoroethanol or hexafluoroisopropanol.     

The chain length of lignan macromolecule from flaxseed hulls is determined by the incorporation of coumaric acid glucosides and ferulic acid glucosides

Lignan macromolecule from flaxseed hulls is composed of secoisolariciresinol diglucoside (SDG) and herbacetin diglucoside (HDG) moieties ester-linked by 3-hydroxy-3-methylglutaric acid (HMGA), and of p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) moieties ester-linked directly to SDG. The linker molecule HMGA was found to account for 11% (w/w) of the lignan macromolecule. Based on the extinction coefficients and RP-HPLC data, it was determined that SDG contributes for 62.0% (w/w) to the lignan macromolecule, while CouAG, FeAG, and HDG contribute for 12.2, 9.0, and 5.7% (w/w), respectively.Analysis of fractions of lignan macromolecule showed that the higher the molecular mass, the higher the proportion of SDG was. An inverse relation between the molecular mass and the proportion (%) CouAG + FeAG was found. Together with the structural information of oligomers of lignan macromolecule obtained after partial saponification, it is hypothesized that the amount of CouAG + FeAG present during biosynthesis determines the chain length of lignan macromolecule.Furthermore, the chain length was estimated from a model describing lignan macromolecule based on structural and compositional data. The average chain length of the lignan macromolceule was calculated to be three SDG moieties with CouAG or FeAG at each of the terminal positions, with a variation between one and seven SDG moieties.     

Stereochemistry of peptides containing a thioacyl group

Endothiopeptides are oligopeptides in which one or more oxoacyl moieties in peptide groups are replaced with thioacyl moieties. The stereochemical consequences of this replacement are discussed and compared with results from X-ray crystallographic determinations of small oligopeptides containing one or two thiopeptide groups. The analysis shows that the conformations normally observed around C alpha carbon atoms in proteins are also stereochemically favourable for peptides in which a thiopeptide group has been introduced. Limitations are only present in cases where the thiopeptide is part of structural elements like helices and beta-sheets. This opens the possibility of using one or more thiopeptide groups as functional modifiers of biologically active oligopeptide and protein molecules.     

The structure of Artemia sp. haemoglobin. Cleavage of the native molecules into functional units by limited subtilisin digestion.

Limited subtilisin digestion of the high-Mr haemoglobin of the crustacean Artemia sp. results in a series of fragments that are multiples of Mr 16000. Properties such as amino acid composition, iron content, absorption and c.d. spectra of the 16000-Mr functional units strongly resemble those of the intact haemoglobin molecules. The 16000-Mr functional units can bind O2 in a non-co-operative way. They thus represent the structural units from which the globin chains are built up.     

The MPB83 antigen from Mycobacterium bovis contains O-linked mannose and (1-->3)-mannobiose moieties

Mycobacterium tuberculosis and Mycobacterium bovis, the causative agents of human and bovine tuberculosis, have been reported to express a range of surface and secreted glycoproteins, although only one of these has been subjected to detailed structural analysis. We describe the use of a genetic system, in conjunction with lectin binding, to characterize the points of attachment of carbohydrate moieties to the polypeptide backbone of a second mycobacterial glycoprotein, antigen MPB83 from M. bovis. Biochemical and structural analysis of the native MPB83 protein and derived peptides demonstrated the presence of 3 mannose units attached to two threonine residues. Mannose residues were joined by a (1 --> 3) linkage, in contrast to the (1 --> 2) linkage previously observed in antigen MPT32 from M. tuberculosis and the (1 --> 2) and (1 --> 6) linkages in other mycobacterial glycolipids and polysaccharides. The identification of glycosylated antigens within the M. tuberculosis complex raises the possibility that the carbohydrate moiety of these glycoproteins might be involved in pathogenesis, either by interaction with mannose receptors on host cells, or as targets or modulators of the cell-mediated immune response. Given such a possibility characterization of mycobacterial glycoproteins is a step toward understanding their functional role and elucidating the mechanisms of mycobacterial glycosylation.     

The role of scaffold attachment regions in the structural and functional organization of plant chromatin

Studies on nuclear scaffolds and scaffold attachment regions (SARs) have recently been extended to different plant species and indicate that SARs are involved in the structural and functional organization of the plant genome, as is the case for other eukaryotes. One type of SAR seems to delimit structural chromatin loops and may also border functional units of gene expression and DNA replication. Another group of SARs map close to regulatory elements and may be directly involved in gene expression. In this overview, we summarize the structural and functional properties of plant SARs in comparison with those of SARs from animals and yeast.